highly conserved salt bridge stabilizes a proteinase k subfamily enzyme aqualysin i from thermus aquaticus yt 1

To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to

O R I G I N A L A R T I C L E Open Access

Highly conserved salt bridge stabilizes a proteinase

aquaticus YT-1

Masayoshi Sakaguchi1*†, Kanae Osaku1†, Susumu Maejima1, Nao Ohno1, Yasusato Sugahara1, Fumitaka Oyama1 and Masao Kawakita1,2

Abstract

The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp PA-44, have six and seven salt bridges, respectively To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12 Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme These findings will further our understanding of the factors involved in stabilizing protein structure

Keywords: Serine protease; Subtilase; Proteinase K subfamily; Salt bridge; Thermal stability

Introduction

The molecular bases of protein adaptation to high and

low temperatures are interesting from both basic and

practical standpoints, as knowledge regarding these factors

would enable the construction of genetically engineered

proteins that could function under a variety of conditions

Psychrophilic and mesophilic enzymes are used in

bio-technological applications requiring high activity at mild

temperatures or quick heat-inactivation at moderate

tem-peratures Thermophilic and hyperthermophilic enzymes

have major biotechnological advantages over mesophilic

and psychrophilic enzymes because of their high activities

at higher temperatures and substrate concentrations as

well as their resistance to chemical denaturants Thus

far, various intramolecular interactions, including ionic interactions, hydrogen bonding and hydrophobic inter-actions, are assumed to make important contributions

to the stability and maintenance of enzyme structure as well as the catalytic functions; however, their contributions have not been fully defined in individual cases In add-ition, the comparative structural analysis of psychrophilic, mesophilic and thermophilic enzymes indicated that each protein family adopts a different structural strategy to adapt to different temperature ranges (Siezen & Leunissen 1997; Struvay & Feller 2012) To understand the thermal adaptation strategy of proteins, comparative studies among members of a protein family using site-directed mutagen-esis as well as laboratory evolution via random mutagenmutagen-esis using error-prone PCR will provide valuable information According to the MEROPS peptidase database (http:// merops.sanger.ac.uk/), subtilisin-like protease (subtilase) superfamily members are classified as the S8 subfamily in the serine protease superfamily These proteins exhibit a

* Correspondence: bt11532@ns.kogakuin.ac.jp

†Equal contributors

1

Department of Applied Chemistry, Kogakuin University, 2,665-1 Nakano-cho,

Hachioji, Tokyo 192-0015, Japan

Full list of author information is available at the end of the article

© 2014 Sakaguchi et al.; licensee Springer This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction

highly conserved arrangement of amino acids in the active

site and have very similar overall structures consisting

of anα/β protein scaffold Nevertheless, their temperature

stability profiles differ widely, and they may be

psychro-philic, mesopsychro-philic, thermophilic or hyperthermophilic

depending on the characteristics of the organisms from

which they are derived Because of these characteristics,

they appear to be suitable for comparative studies to

elucidate the basis of structure-function relationships

Aqualysin I (AQN) is an alkaline serine-protease produced

by the Gram-negative thermophilic bacterium Thermus

aquaticus YT-1 (Matsuzawa et al 1983; Matsuzawa et al

1988) Based on an analysis of sequence homology, AQN

is classified into the proteinase K subfamily, which

consists of a group of Gram-negative bacteria-derived

proteinases within the subtilase superfamily (Siezen &

Leunissen 1997) In our previous study, we demonstrated

that Pro residues in the surface loops of AQN in the

N-terminal region contribute significantly to its

thermo-philicity, and one of two disulfide bonds in AQN is more

important for the catalytic activity and conformational

stability of AQN than the other (Sakaguchi et al 2007;

Sakaguchi et al 2008b) These results are consistent with

those reported for a subtilisin-like serine protease from

Vibrio sp., VPR, which is a psychrophilic counterpart of

AQN in the proteinase K subfamily (Kristjánsson et al

1999; Arnórsdóttir et al 2002) It was found that the

introduction of Pro residues into VPR at positions

cor-responding to those in AQN could improve its thermal

stability (Arnórsdóttir et al 2009)

To enhance the thermal stability of a protein, a common

strategy is to introduce more favorable surface

charge-charge interactions However, the role of salt bridges in the

stabilization of proteins remains controversial

Thermo-philic proteins have an increased number of salt bridges

compared with their mesophilic homologues (Kumar et al

2000; Szilágyi & Závodszky 2000; Vogt et al 1997) In

VPR, a psychrophilic enzyme, seven salt bridges

(Arg10-Asp183, Arg14-Asp274, Asp56-Arg95, Asp59-Arg95,

Asp138-Arg169, Arg185-Asp260 and Glu236-Arg252)

have been identified in the known structure of the

enzyme (PDB accession number: 1SH7; (Arnórsdóttir

et al 2005)) However, AQN has six salt bridges

(Arg12-Asp183, Asp17-Arg259, Arg31-Asp237, Arg43-Asp212,

Asp58-Arg95 and Asp138-Arg169) in its structure (4DZT;

(Green et al 1993)) (Table 1) Both enzymes have a similar

number of salt bridges; however, their thermal stabilities

are quite different The VPR N15D mutant, in which an

Asp residue is substituted for Asn15 to form a new salt

bridge (Asp15-Lys257) at the position corresponding to

the Asp17-Arg259 salt bridge in AQN, exhibited increased

thermal stability due to the incorporation of a new salt

bridge; the thermal stability of the enzyme did not

in-crease further in the double mutant VPR N15D/K257R

(Sigurdardóttir et al 2009) Inversely, the deletion of the Asp17-Arg259 salt bridge in the AQN D17N mutant resulted in reduced thermal stability compared to the wild-type enzyme without exerting a significant effect

on the kinetic parameters of the hydrolysis reaction (Arnórsdóttir et al 2011) These results may imply that salt bridges at appropriate locations play a vital role in the thermal stability of serine proteases, particularly those of the proteinase K subfamily To help clarify this complicated issue and to further extend our understand-ing of the molecular basis of proteinase K-related enzyme stabilization, we aimed to examine the role of salt bridges

in AQN by site-directed mutagenesis

Materials and methods

Strains and growth medium

E coli TG1 was used as the expression host, and E coli DH5α (TOYOBO, Osaka, Japan) and MV1184 (TAKARA BIO INC., Shiga, Japan) were used as the genetic en-gineering hosts LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 1% NaCl, pH 7.0) was used The solid medium contained Bacto-agar (1.5%) Ampicillin

medium as needed

Genetic engineering and chemical reagents

Genetic engineering experiments were performed accord-ing to the procedure described by Sambrook and Russell (Sambrook & Russell 2012) The enzymes used for genetic engineering were purchased from TAKARA BIO and used according to the manufacturer’s instructions Bacto-tryptone and Bacto-yeast extract were purchased from Becton Dickinson (Franklin Lakes, NJ, USA) Other re-agents used were of the highest quality available and were obtained from Wako Pure Chemicals (Tokyo, Japan) and Sigma-Aldrich (St Louis, MO, USA), unless otherwise specified

Plasmid construction

The plasmid pMAQΔc2, which was designed to express wild-type AQN as a fusion protein with maltose binding protein (MBP), was constructed based on pAQNΔC105 and pMAL plasmids (New England Biolabs, Ipswich, MA, USA) as described previously (Sakaguchi et al 2008a)

To construct plasmids with a mutated aqualysin I gene, site-directed mutagenesis was performed following the ODA-PCR method (Mutan®-Super express Km; TAKARA BIO) using pMAQΔc2 as a template The oligonucleotide primers (Sigma-Aldrich Life Science, Hokkaido, Japan) used for site-directed mutagenesis are shown in Table 2 The fragments containing either of the mutations listed

in the table were inserted into the expression vector, pMAQΔc2 The names of the mutant plasmids are pro-vided in the third column of Table 2 (D17N, etc.) The

nucleotide sequences around the mutation sites as well

as other parts of the gene were confirmed by DNA

sequencing using an Applied Biosystems 3130 Genetic

Analyzer (Applied Biosystems, Foster City, CA, USA)

Purification and activity measurement of the wild-type

enzyme and its mutants

After induction by isopropyl β-D-thiogalactopyranoside

(IPTG) at OD660= 0.8, the transformants were further

cultivated overnight in LB medium The cells were

harvested by centrifugation and subsequently sonicated,

and the crude extract was subjected to heat treatment,

hydrophobic chromatography (Butyl Sepharose; GE

Healthcare, Buckinghamshire, UK) and cation exchange

chromatography (Resource S; GE Healthcare) as described

previously (Sakaguchi et al 2008a) The enzymes were

purified to homogeneity to yield a single band on

SDS-polyacrylamide gel electrophoresis (PAGE) after staining

with Coomassie Brilliant Blue R-250 (CBB R-250)

(Laemmli 1970) Prior to SDS-PAGE analysis, the enzymes

were treated with 25 mM phenylmethane sulfonyl fluoride

(PMSF) dissolved in methanol for 30 min to prevent

autolytic degradation To minimize denaturation and autolysis which may occur at higher temperature, the enzyme activity was measured at 40°C with N-succi-nyl-Ala-Ala-Pro-Phe-p-nitroanilide (N-suc-AAPF-pNA, Sigma-Aldrich) as a substrate in 50 mM 2-[4-(2-hydro-xyethyl)-1-piperazynyl]ethanesulfonic acid (HEPES)-NaOH (pH 7.5) buffer containing 1 mM CaCl2 The change in absorbance at 410 nm was continuously monitored, and the activity was estimated usingε410= 8,680 M−1cm−1as

a molar absorption coefficient of p-nitroaniline (4-nitroa-niline) One unit of enzyme was defined as the amount

of enzyme that liberates 1μmole of p-nitroaniline from the substrate in 1 minute The protein concentration was measured using the micro-assay method (Bio-Rad Laboratories, Hercules, CA, USA), which is based on the Bradford method (Bradford 1976), using bovine serum albumin as a standard

Determination of the temperature dependence of the proteolytic activity and heat stability of the wild-type enzyme and its mutants

To examine the temperature dependence of the enzyme

100 mM HEPES-NaOH (pH 7.5) containing 1 mM

410 nm was continuously monitored, and the activity was estimated as described above based on the results

of triplicate experiments

To examine the heat stability, the enzymes were diluted with 20 mM 2-morpholinoethanesulfonic acid (MES)-NaOH buffer (pH 6.0) containing 1 mM CaCl2to yield a

at pH 6.0 to diminish the massive autolysis that would occur under more alkaline condition This enabled us to observe the differential decrease of residual activity among mutants due to structural destabilization during the heat

Table 2 Oligonucleotide primers used for site-directed

mutagenesis

AQN-D58N 5'-GGTAGGCTATAACGCCTTAGGG-3' D58N

AQN-G61D 5'-ACGCCTTAGATGGGAACG-3' G61D

AQN-D138N 5'-CTGCCCTGAACAACGCC-3' D138N

AQN-D183N 5'-ATCTTCCAACGCCCGTG-3' D183N

AQN-D212N 5'-ACACCTCGAACACGGCC-3' D212N

AQN-E237Q 5'-CTTTGTATCTACAGCAAAATCTTC-3' E237Q

AQN-G262D 5'-GCTTTCGGATATCGGATCG-3' G262D

AQN-S277D 5'-CCTGCTCGATTCGGGGAG-3' S277D

Mismatched bases are shown in italic.

Table 1 Amino acids that form salt bridges in AQN, VPR and SPRK

Asp198-Lys254

Arg31-Glu237 Arg43-Asp212

treatment processes The enzyme solution was incubated

for the appropriate time period at 70°C or 80°C and was

subsequently cooled quickly The remaining activity was

determined based on the results of triplicate experiments

using 1 mM N-suc-AAPF-pNA as a substrate at 40°C, as

described above

Kinetic analysis

The initial rates of N-suc-AAPF-pNA hydrolysis induced

by the wild-type enzyme and mutant enzymes were

measured at 40°C in 50 mM HEPES-NaOH (pH 7.5)

containing 1 mM CaCl2as described above The kinetic

Michaelis-Menten kinetic model, and the graphics

soft-ware package DeltaGraph version 6 (Nihon Poladigital

K.K., Tokyo, Japan) was used with non-linear regression

The apparent values of kcat were estimated using a

mo-lecular mass of 28 kDa

Unfolding study of AQN proteins based on circular

dichroism (CD) measurement

CD analysis was carried out to determine the transition

temperature (Tm) and to monitor the unfolding of the

wild-type and mutant enzymes Prior to the CD

mea-surements, purified enzymes were treated with 25 mM

PMSF dissolved in methanol for 30 min to prevent autolytic

degradation during the measurements After complete

inactivation of the protease activity was confirmed, the

samples were dialyzed overnight against 20 mM

MES-NaOH buffer (pH 6.0) containing 1 mM CaCl2and filtered

through a MILEX®-HV filter (0.45μm pore size, Durapore

(PVDF), Merck Millipore Ltd., Carrigtwohill, Ireland) CD

measurements were conducted using a JASCO-725 circular

dichroism spectropolarimeter equipped with PTC-348

Peltier type single cell holder, and the change in ellipticity

at 220 nm was monitored under a constant heating rate

(1°C/min) at temperatures ranging from 40 to 105°C The

melting curves were normalized according to the methods

in the literature (Arnórsdóttir et al 2002), and the melting

temperature (Tm) values of the enzymes were estimated

using a graphics software package, Delta graph The

experiments were performed in duplicate

The unfolding of proteins as a function of time was

observed at a constant temperature (70°C or 80°C) by

CD measurement over a range of 200–250 nm

Mea-surements were performed with a JASCO-725 circular

dichroism spectropolarimeter (JASCO, Tokyo, Japan)

equipped with PTC-348 Peltier type single cell holder,

and the change in ellipticity was monitored at every

5 min over a 30-min period at a constant temperature

Wavelength scans in the range of 200–250 nm were

performed in rectangular quartz cells (JASCO model:

T-11-ES-1) with a path length of 0.1 cm

Results

Mutagenesis of salt bridge-forming residues in AQN and purification of the wild-type enzyme and its mutants

Table 1 lists the salt bridge-forming amino acid residues

in AQN, VPR and Serratia proteinase K-like enzyme (SPRK; (Larsen et al 2006)), which were identified based

on the known structures of AQN (4DZT; (Green et al 1993)), VPR (1SH7; (Arnórsdóttir et al 2005)) and SPRK (2B6N; (Helland et al 2006)), respectively There are three conserved salt bridges in both AQN and VPR (Figure 1a and b) In addition, AQN and VPR have three (Asp17-Arg259, Arg31-Glu237 and Arg43-Asp212) and four (Asp59-Arg95, Arg14-Asp274, Arg185-Asp260 and Glu236-Arg252) intrinsic salt bridges, respectively (Figure 1c, d, e and f ) To examine the role of salt bridges

in AQN, the Asp and Glu residues were replaced with Asn and Gln residues, respectively, to make them incapable of forming salt bridges To introduce new salt bridges in AQN

at the positions at which the VPR-intrinsic salt bridges are located, the residues Gly61, Gly262 and Ser277 were con-verted to Asp residues to make them capable of forming salt bridges with Arg95, Arg185 and Arg16, respectively, which are conserved in both AQN and VPR The mutant AQN constructs were expressed, and the protein products were purified to homogeneity using essentially the same methodology used for the wild-type enzyme

Thermal stability of the wild-type enzyme and its mutants

The remaining activity of the wild-type and mutant enzymes after heat treatment at temperatures of 70°C

or 80°C was determined at 40°C using N-suc-AAPF-pNA

as a substrate; the results are illustrated in Figure 2a-f Figure 2a and b show the time courses of the residual activity at 70°C and 80°C, respectively, for the D17N, D212N and E237Q mutants lacking a salt bridge specific

to AQN At 70°C, the activity of E237Q and D17N decreased more rapidly than that of the wild-type enzyme, indicating that the mutants are less stable at 70°C D212N behaved similarly to the wild-type enzyme at 70°C; however,

at 80°C, it was inactivated as rapidly as E237Q and D17N (Figure 2a and b) These results indicate that the AQN-intrinsic salt bridges Arg31-Glu237, Asp17-Arg259 and Arg43-Asp212 contribute significantly to the maintenance

of active enzyme structures at temperatures above 70-80°C Disruption of the salt bridges common to AQN and VPR did not yield a common outcome For example, the residual activity of D183N declined rapidly, with a half-life of approximately 30 min at both 70°C and 80°C, whereas the activities of D58N and D138N were almost indistinguishable from that of the wild-type enzyme at both temperatures (Figure 2c and d) The above results indicate that the Arg12-Asp183 salt bridge is important for conferring structural stability to proteinase K subfamily enzymes, although the other two common salt bridges

are not significantly involved in thermal stabilization.

Additional salt bridges were introduced at the positions

where VPR-intrinsic salt bridges are located in mutants

G61D, G262D and S277D The inactivation time courses

of the three mutants were similar to that of the wild-type

enzyme at 70°C and 80°C In fact, S277D was slightly less

stable than the wild-type enzyme at both 70°C and 80°C

(Figure 2e and f )

Temperature dependence of wild-type and mutant enzyme

activity

Figures 3a-c compares the temperature dependence of

wild-type and mutant enzyme activity All mutants

displayed similar temperature-activity profiles over a range of 30-90°C Disrupting a salt bridge or introdu-cing a potential salt bridge-forming mutation did not lead to an extensive reduction of enzyme activity In fact, the enzyme activity of the mutants tended to be slightly higher than that of the wild-type enzyme espe-cially at higher temperatures D17N showed the high-est activity among the mutants thigh-ested: its activity was about 1.5-fold as high as that of the wild-type enzyme

at 90°C The optimum temperature was not altered significantly by the mutations, and every mutant showed full activity at approximately 90°C, similar to the wild-type enzyme

D58-R95

D138-R169

R12-D183

D56 R95

D138-R169

Ca2 Ca1

Ca1

Ca3

D17-R259

R43-D212

D59-R95

R185-D262

R14-D274

Ca1

Ca3

Ca3

Ca2 Ca1

E236-R252

R185-D262 Ca3

Ca1

R14-D274

D17-R259

R31-E237

Ca1

Ca3

Figure 1 Crystal structures of AQN (a, c, e; 4DZT) and VPR (b, d, f; 1SH7) (a, b) Common salt bridge positions, (c, d) intrinsic salt bridge positions, (e, f) intrinsic salt bridge positions rotated approximately 90 degrees horizontally from (c, d) The salt bridges of interest are indicated

by residue numbers and are represented as stick models Calcium ions (Ca1, Ca2 and Ca3) are represented as pale green spheres.

Figure 2 Heat stability of the wild-type and mutant enzymes Heat stability was determined as described in the Materials and methods section The remaining activities after heat treatment at 70°C (a, c and e) or 80°C (b, d and f) for the indicated times relative to the activity before heat treatment are plotted for each mutant (a, b) mutants lacking an intrinsic salt bridge, (c, d) mutants lacking a common salt bridge, (e, f) mutants in which a salt bridge may have been introduced.

Kinetic analysis of wild-type and mutant enzymes

The kinetic parameters of wild-type and mutant enzymes

in the presence of a synthetic substrate,

N-suc-AAPF-pNA, at 40°C are shown in Table 3 The Kmvalues of the

mutants were similar to that of the wild-type enzyme,

except that the Km value of D212N was slightly higher

than that of the wild-type enzyme and the other mutants

The kcatvalues of the mutants were in the range of

enzyme value This result suggests that depletion of a salt

bridge or introduction of a new salt bridge to these sites

does not profoundly affect the integrity of the structure of

AQN at 40°C Structural integrities of mutants were also

confirmed below by the similarity between CD spectra of

wild type and mutant enzymes (see Figure 4)

Unfolding study on AQN proteins based on CD

measurements

To analyze the unfolding process of active AQN mutants

at a fixed temperature, changes in the CD spectra of

the wild-type enzyme, D17N, E237Q, D212N or D183N

as a function of time were recorded at 70°C and 80°C

Figure 4a and b show the CD spectra of the wild-type

enzyme during the 30-min incubations at 70°C and 80°C,

respectively The spectrum was not significantly changed

during the 30-min incubation at 70°C However, at 80°C, the ellipticity at 222 nm was slightly but significantly de-creased in the first 5 min, and it subsequently remained unchanged for up to 30 min Figure 4c and d show the

CD profiles of D17N during the 30-min incubations at 70°C and 80°C, respectively The change in ellipticity at

222 nm at 70°C followed a time course that was very similar to that of the wild-type enzyme at 80°C However,

Figure 3 Temperature-dependence of the activity of the wild-type and mutant enzymes The temperature-dependence of the activity was measured as described in the Materials and methods section (a) mutants lacking an intrinsic salt bridge, (b) mutants lacking a common salt bridge, (c) mutants in which a salt bridge may have been introduced.

Table 3 Kinetic parameters of the wild-type and mutant enzymes1

Enzyme k cat (s−1) K m (mM) k cat / K m (mM−1s−1) Wild-type 91.6 ± 2.75 0.79 ± 0.04 116

D212N 75.2 ± 3.13 1.10 ± 0.10 68.6 E237Q 96.1 ± 0.98 0.91 ± 0.03 105 D58N 68.5 ± 1.37 0.99 ± 0.07 70.0 D138N 59.5 ± 6.14 0.77 ± 0.01 77.3 D183N 53.0 ± 3.03 0.74 ± 0.05 71.6 G61D 47.2 ± 1.77 0.82 ± 0.13 63.6 G262D 61.3 ± 1.86 0.93 ± 0.15 66.2 S277D 73.6 ± 5.51 0.80 ± 0.12 92.5

1

Parameters were determined at 40°C with N-suc-AAPF-pNA as a substrate The experiments were performed in either duplicate or triplicate.

at 80°C, the initial change at 5 min was slightly more

pro-nounced and there was a gradual decrease in ellipticity

that continued for up to 30 min in the D17N mutant

The change in the CD spectrum of E237Q was similar

to that of D17N during incubation at both 70°C and 80°C

(Figure 4e and f ) These results indicated that the

second-ary structure contents of D17N and E237Q did not show

major decreases for 30 min at 70°C The change in the CD

profile of D212N was very similar to that of D17N at 70°C

(Figure 4g) However, at 80°C, the ellipticity of D212N in

the 200–240 nm range continued to decrease for up to

30 min in parallel with the rapid inactivation of this

mutant at 80°C (Figures 4h and 2b) The change in the

CD profile of D183N at 70°C is shown in Figure 4i The

peak at 222 nm gradually decreased as a function of time

down to 40% of the value before treatment, indicating that

more than half of the secondary structures of D183N were destroyed A comparable change in the ellipticity

of D212N occurred only at 80°C These results suggest that a salt bridge involving Asp183 plays a significant role in maintaining the structure of AQN This may also

be true of other members of the proteinase K subfamily,

as the salt bridge involving Asp183 is conserved among these enzymes

The denaturation curve of the PMSF-treated D183N mutant as monitored by the change in the ellipticity at

220 nm is shown in Additional file1: Figure S1 The

consistent with the extensive decrease of 222 nm peak intensity observed in Figure 4i Denaturation of other mutants as well as the wild type enzyme apparently occurred at much higher temperature range than D183N

a

c

d

i

b

f e

g

h

Figure 4 The change in CD spectra of the wild-type and mutant enzymes over a range of 0 –30 min at a constant temperature (a) Wild-type enzyme at 70°C, (b) wild-type enzyme at 80°C, (c) D17N at 70°C, (d) D17N at 80°C, (e) E237Q at 70°C, (f) E237Q at 80°C, (g), D212N at 70°C, (h), D212N at 80°C, (i) D183N at 70°C The experimental conditions are described in the Materials and methods section.

mutant, but we could not obtain reliable estimates for

their Tmvalues It should be noted that the change in CD

spectrum gradually proceeded over 30 min at constant

temperature as shown in Figure 4, while measurement

of Tm was carried out at a heating rate of 1°C/min It is

possible that the rate of protein unfolding did not catch

up with the elevation of the temperature, and this

might give apparent Tmhigher than true Tm

Discussion

Subtilases, a group of serine proteases in the subtilisin

superfamily, are composed of approximately 275 amino

acid residues Mutation studies on more than 50% of the

amino acid residues in their primary structure have been described in the literature (Bryan 2000) Various factors appear to make complex contributions to subtilase stabil-ity There are six subfamilies in the subtilisin superfamily, and it is possible that the mechanism by which thermal stability is conferred may differ from one subfamily to another In this report, we investigated the roles of salt bridges in the thermal stabilization of AQN, a proteinase

K subfamily member

Regarding the salt bridges intrinsic to AQN (Asp17-Arg259, Arg31-Glu237 and Arg43-Asp212), the kcatvalues

of D17N and E237Q at 40°C and the activities at elevated temperatures toward a synthetic substrate were slightly

Figure 5 Alignment of the primary structures of proteinase K subfamily enzymes and subtilisin BPN ’ AQN, aqualysin I; VPR, protease from Vibrio sp PA-44; SPRK, proteinase K-like enzyme from Serratia sp.; PK, proteinase K from Tritirachium album Limber; BPN, subtilisin BPN’ from Bacillus amyloliquefaciens “#” denotes the catalytic residues Asp, His and Ser The positions of common (closed circles), AQN-intrinsic (open arrowheads) and VPR-intrinsic (closed arrowheads) salt bridges are represented.

increased compared to that of the wild-type enzyme;

however, these mutants were apparently less

thermo-stable than the wild-type enzyme during heat treatment

experiments These results suggest that the decline of

the residual activities of the mutant enzymes during

prolonged incubation at high temperatures might be

caused in part by extensive autolysis under conditions

in which these enzymes exhibit higher activity than the

wild-type enzyme However, it should also be noted

that examination by CD spectrometry indicated that

D17N and E237Q showed a small change in ellipticity at

222 nm in the first 5 min at 70°C, although the ellipticity

remained unchanged over the course of the subsequent

incubation for up to 30 min (Figure 4c and e) These

results suggest that incubation at 70°C may affect regions

of the D17N and E237Q mutants that are not rich in

secondary structures, including loop regions, but that

the structural perturbation due to the D17N or E237Q

mutation may destabilize the active site conformation

during a prolonged incubation at 70°C and above This

result agrees with a previous report indicating that the D17N mutant exhibited reduced thermal stability com-pared to the wild-type enzyme (Arnórsdóttir et al 2011) Recently, Jakob et al reported an intensive analysis of the roles of charged amino acid residues in a Bacillus gibsonii subtilisin protease, BgAP, using site-directed mutagenesis BgAP Q230E showed increased thermal re-sistance compared to wild-type BgAP (Jakob et al 2013) This result is consistent with our data on E237Q Glu237

of AQN corresponds to Gln230 of BgAP, and disruption

of a salt bridge in E237Q resulted in a rapid decrease of activity during incubation at 70°C and 80°C

The stability of D212N was similar to that of the wild-type enzyme at 70°C; however, it was inactivated rapidly

at 80°C (Figure 2a and b) This result is consistent with the results of the CD spectrometry analysis showing that the secondary structure content was rapidly decreased as

a function of time at 80°C The inactivation mechanism of D212N at 80°C may be different from that of D17N and E237Q at 70°C

R12-D183

R10-D183

Ca3

Ca1

Ca3

Ca1

Figure 6 Crystal structures of AQN (a, 4DZT) and VPR (b, 1SH7) The positions of the salt bridges, (a) Arg12-Asp183 of AQN and (b) Arg10-Asp183 of VPR, are represented as stick models Calcium ions (Ca1 and Ca3) are represented as pale green spheres.

R12-D183

D17-R259

C163-C199

C163-C199

D17-R259

R12-D183

P5 P5

P7 P7

C67-C99

R31-E237

R43-D212

Figure 7 Crystal structure of AQN (4DZT) with residues contributing to enzyme stabilization (a) The residues contributing to the salt bridges Arg12-Asp183, Asp17-Arg259, Arg31-Glu237 and Arg43-Asp212 and the disulfide bridges Cys67-Cys99 and Cys163-Cys199 are indicated and are represented as stick models Calcium ions are represented as pale green spheres The catalytic residues are represented as blue stick models (b) The structure is rotated approximately 90 degrees horizontally from (a).